Human fungal infections are serious and often life-threatening, particularly for immunocompromised patients. Immunocompromised patients provide a great challenge to modern health care delivery. For example, the mean survival time of AIDS patients with cryptococcal infections is 8.4 months (NIH meeting; Medical Mycology, June, 1993). These infections are often the result of opportunistic fungi that are usually asymptomatic commensals, as described by M. Shepard; R. Poulter & P. Sullivan: "Candida albicans: Biology, Genetics, and Pathogenicity", Ann. Rev. Microbiol. Vol. 39 (1985) pp. 579-614.
Immune deficiencies, which are often caused by antineoplastic chemotherapy, organ transplants, congenital defects, leukemia, Hodgkin's disease, diabetes, and HIV infection render an immunocompromised host susceptible to a large number and variety of neoplastic, protozoal, viral, bacterial, and fungal diseases. Of these, bacterial, viral and fungal infections result in the greatest mortality. During the last three decades there has been a dramatic increase in the frequency of fungal infections, especially disseminated systemic mycoses in immunodeficient hosts. As previously mentioned, mycoses in compromised hosts are mainly the result of opportunistic infections by organisms that are normally harmless asymptomatic commensals, which can be, under certain conditions, pathogenic. Fungi of particular importance include: Cryptococcus, Candida, Coccidioides, Histoplasma, Sporothrix, and Aspergillus; of these Candida infections are the most common. Candidiasis has a large number of clinical presentations ranging from cutaneous to disseminated systemic infections and includes oral thrush, bronchitis, meningitis, septicemia, asthma, gastritis, uveitis and endocarditis. Additionally, Pneumocystis carnii is the leading cause of deaths in AIDS patients and recent data based on 16S-like RNA sequence analysis reveal that Pneumocystis carnii is related to fungi.
It is known that the treatment of mycotic infections is difficult due to a lack of effective antifungal antibiotics. See, for example, Ringel, S.; "New Antifungal Agents for the Systemic Mycoses", Mycopath. Vol. 109 (1990) pp. 75-87; Walsh, T.; P. Jarosinski & R. Fromtherling: "Increasing Usage of Systemic Antifungal Agents", Diagn. Microbial. Infections, Vol. 13, (1990) pp. 37-40; Medoff, G.; J Brajtburg, J. Boland & G. Kobayashi: "Antifungal Agents Useful in Therapy of Systemic Fungal Infections", Ann. Rev. Pharm. Tox. Vol. 23 (1983) pp. 303-330. Even after 29 years of use Amphotericin B (a polyene) is still the drug of choice to treat systemic fungal infections. See Ryley, J.; R. Wilson, M. Gravestock & P. Poyser: "Experimental Approaches to Antifungal Chemotherapy", Adv. Pharm. Chemother. Vol. 18 (1981) pp.49-177; Gallis, H.; R.Drew & W. Pickard: "30 Years of Clinical Experience, Rev. Inf. Dis. Vol. 12 (1990) pp. 308-329.
The apparent mode of action of Amphotericin B (AmB) is to complex with membrane sterols, resulting in membrane distortion and leakage of intracellular contents. In addition, AmB is an immunostimulant. However, AmB is very toxic to human cells and AmB therapy is fraught with side effects which include renal dysfunction, fever, chills, hypotension and even cardiac failure. In spite of toxicity and problems with formulation (AmB is not orally active but must be administered intravenously), AmB is the most used systemic antifungal agent.
Other treatment options include imidazoles (for example Ketoconazole, miconazole), which inhibit fungal growth by inhibiting the C-14 demethylation step in sterol biosynthesis. Although are orally active, they are not recommended for use in the treatment of systemic infections in immunocompromised patients. Ryley, J.; R. Wilson, M. Gravestock & P. Poyser: "Experimental Approaches to Antifungal Chemotherapy", Adv. Pharm. Chemother. Vol. 18 (1981) pp.49-177. Still other antifungal agents exist, for example flucytosine, Cilofungin, Papulacandin B, Aculeacin A, yet they are limited by either narrow spectrum of activity or by toxicity or both. The limitations of the prior art indicate a need for new, effective antifungal antibiotics that eliminate the side effects of those antibiotics available today.
The key to developing effective antifungal therapeutics lies in targeting fungal-specific enzymes or molecules. One such target, perhaps the most unique, is the fungal cell wall. In studying fungal cells, it is noted that the most striking difference between fungal cells and human cells is that fungal cells are encased in a wall which protects them from an osmotically and immunologically hostile external environment. The fungal cell wall relays signals for invasion and infection of a likely plant, animal or human host. The cell wall of fungi has a complex composition and structure and has been the subject of several reviews. See, for example: Ruiz-Herrera, J.: "Fungal Cell Wall: Structure, Synthesis and Assembly, CRC Press. FI., 1992; Gooday, G & N. Gow: "Enzymology of Tip Growth in Fungi. Tip Growth in Plant and Fungal Cells", I. B. Health, Ed. Acad. Press, 1990; Wessels, J. G. H.: "Wall Growth, Protein Excretion and Morphogenesis in Fungi, New Phytol. Vol. 123 (1993) pp. 397-413.
In general, human pathogenic fungi, in both yeast and filamentous forms, contain chitin (G1cNAc polymer), (1,3).beta.-glucan, other glucans (some even contain cellulose), peptides, lipids, and a small amount of unknown material. Fungal cell walls, when fixed and viewed by standard electron microscopic techniques, appear trilayered and roughly 150-250 nm thick. For filamentous fungi, growth and cell-wall assembly occur only at each hyphal apex. Ruiz-Herrera,J. L. "Fungal Cell Wall: Structure, Synthesis and Assembly", CRC Press FI., 1992; Wessels, J. G. H.: "Wall Growth, Protein Excretion and Morphogenesis in Fungi", New Phytol. Vol. 123 (1993) pp. 397-413. Normal cell-wall assembly is essential for growth and viability of a large number of fungi. This proposition has been convincingly shown using cell-wall acting drugs and by the analysis of mutants defective in key wall-assembly steps. See Scott, W. A.: "Biochemical Genetics of Morphogenesis in Neurospora", Ann. Review Microbiol. Vol. 30 (1976) pp. 85-104; Shaw, J.;P. Mol, B. Bowers, D. Silverman, M. Vadiviesco, A, Duran & E. Cabib: "The Function of Chitin Synthases 2 and 3 in the Saccharomyces cerevisiae Cell Cycle", J. Cell Biol. Vol. 114 (1991) pp. 111-123.
There are a large number of single-gene mutants of several fungi that result in abnormal cell-wall assembly and morphogenesis. For example, in the filamentous Ascomycete, Neurospora crassa, there are over 150 single-gene morphological mutants. However, dissecting cell-wall assembly by examining morphological mutants has not proven feasible for it is likely that many of these mutants produce defective enzymes involved in intermediary metabolism. As an example, col-1 is glc-6-P dehydrogenase defective. It has proven nearly impossible to relate directly a deficit in intermediary metabolism to cell-wall assembly. It appears that subtle changes in intracellular metabolite concentrations have dramatic effects on wall assembly and resulting hyphal morphology.
Thus far only three enzyme activities have unequivocally been shown to be directly involved in and essential for cell-wall assembly, namely chitin synthase, (1,6).beta.-glucan synthase and (1,3).beta.-glucan synthase. See Taft, C. & C. Selitrennikoff: "LY121019 Inhibits Neurospora crassa growth and (1,3).beta.-D-glucan synthase. J. Antibiotics, Vol. 41 (1988) pp. 697-701; Romer T. & H. Bussey: "Yeast .beta.-glucan synthase: KRE6 Encodes a Predicted Type Membrane Protein Required for Glucan Synthase in Vivo and for Glucan Synthase Activity in Vitro", Proc. Natl. Acad. Sci. USA vol. 88 (1991) pp. 11295-11299; Roemer, T.; S. Delaney & H. Bussey: "SKN 1 and KRE6 Define a Pair of Functional Homologs Encoding Punative Membrane Proteins Involved in .beta.-Glucan Synthesis", Molec. Cell. Biol. Vol. 13 (1993) pp. 4039-4048. Since mammalian cells lack chitin, (1,6).beta.-glucan synthase and (1,3).beta.-glucan, the fungal pathways for their syntheses are attractive targets for antifungal drugs. Chitin synthase as a target for antifungal drugs has been reviewed. Further, the showing that Pneumocystis carinli pneumonia was treated successfully with (1,3).beta.-glucan synthase inhibitors has greatly stimulated interest in the search for new enzyme inhibitors. See Schmatz, D. et al. "Treatment of Pneumocystis carnii Pneumonia with (1,3).beta.-glucan Synthase Inhibitors", Proc. Natl. Acad. Sci. USA Vol. 87 (1990) pp. 4039-4048.
To date, all the information available shows that (1,3).beta.-glucan synthase is the important enzyme activity for fungal cell-wall and development. It is shown in the prior art that (1,3).beta.-glucan synthase activity is required for normal cell-wall assembly, growth, and development for a large number of fungi, including species of Candida, Aspergillus, and Neurospora. See Wessels, J. G. H.: "Wall Growth, Protein Excretion and Morphogenesis in Fungi, New Phytol. Vol. 123 (1993) pp.397-413; Gorgee, R.: D. Zeckner, L. Ellis, A. Thakkar & L. Howard: "In vitro and in vivo anti-Candida Activity and Toxicology of LY121019", J. Antibiotics, Vol. 37 (1984) pp. 1054-1065; Bozzola, J.; R. Mehta, L. Nisbet & J. Valenta: "The Effect of Aculeacin A and Papulacandin B on Morphology and Cell Wall Ultrastructure in Candida albicans", Can. J. Microbiol. Vol. 30 (1984) pp. 857-863; Miyata, M.; T. Kanbe & K. Tanaka: "Morphological Alterations of the Fission Yeast Schizosaccharomyces pombe in the Presence of Aculeacin A: Spherical Wall Formation, J. Gen. Microbiol. Vol. 131 (1985) pp. 611-621; Perez, P.; Garcia-Ascha & A. Duran: "Effect of Papulacandin B on the Cell Wall and Growth of Geotrichum Lactis, J. Gen. Microbiol. Vol. 129 (1983) pp. 245-250; Perez, P.; R. Varona, I Garcia-Acha & A. Duran: "Effect of Papulacandin F and Aculeacin A on (1,3)-.beta. glucan synthase from Geotrichum lactis", FEBS. Lett. 129, pp.249-252, 1981; Kopecka, M.: "Lysis of Growing Cells of Saccharomyces cerevisiae Induced by Papulacandin B", Folia Microbiol. Vol. 29 (1984) pp. 115-119; Taft, C.; T. Stark & C. Selitrennikoff: "Cilofungin (LY121019) Inhibits Candida albicans (1,3)-.beta.-glucan Synthase Activity, Antimicrobial Agents Cheomother", Vol. 32, pp.1901-1903, 1988; and Phelps, P; T. Stark & C. Selitrennikoff: "Cell-wall Assembly of Neurospora crassa: Isolation and Analysis of Cell-Wall-Less Mutants", Current Microbiol Vol. 2 (1990) pp. 233-242. In each case, when the functioning of (1,3).beta.-glucan synthase was reduced either by drug treatment or by mutations that alter the level of substrate or enzyme activity, resulting cell-wall assembly and morphogenesis were abnormal, that is, cell walls either grew poorly or lysed.
It is known that (1,3).beta.-D-glucan synthase (E.C.2.4.1.34 UDP-glucose: 1,3-.beta.-D-glucan 3-.beta.-glucosyl transferase) catalyzes the polymerization of glucose (1-3!-.beta.-linkages) using UDP-glucose as substrate (the K.sub.m values range between 0.1 mM to 5 mM depending on source of the enzyme), although it has been reported in one instance that GDP-glucose was the preferred substrate. There is little known, however, concerning the structural requirements and order of binding for catalysts. By analogy with other glucosyl transferases, it is likely that the polymerization reaction involves the substrate acting as an electrophile while the glucan chain participates as a nucleophile. Enzyme activity is particulate and localized to the plasma membrane, while the in vitro pH optimum is typically between pH 7 and pH 8. Enzyme activity does not require a divalent metal ion, does not use a lipid-linked intermediate, and activity is not zymogenic, i.e. not proteolytically activated, and enzyme activity does not require a primer. A GTP-binding protein seems to play an important regulatory role and can be dissociated from "core" enzyme activity. Further, .beta.-linked disaccharides are activators; inhibitors include uridine nucleotides, Neopeptins, Aculeacin A, Echinocandin B, gluconolactone, Papulacandin B, sorbose and Cilofungin. The site for substrate hydrolysis is cytoplasmic facing and the resulting glucan polymer is vectorially synthesized to the extracytoplasmic face of the plasma membrane. (1,3).beta.-glucan synthase is found in essentially all groups of fungi and is also present in plants (callose synthase).
Although detailed comparisons between fungal and plant glucan synthase have not been done, recent results show that enzyme activity from fungi and plants is multimeric. However, differences between (1,3).beta.-glucan synthase of fungi and plants are emerging. For example, plant (1,3).beta.-glucan synthase activity requires Ca.sup.2+ and Mg.sup.2+ but is not activated by GTP; fungal (1,3).beta.-glucan synthase requires neither ion and is activated by GTP.
One approach to the study of cell-wall assembly has been the genetic dissection of polymer synthesis. This has involved several strategies, one of which has been to characterize mutants and genes conferring resistance to killer toxins that bind .beta.-glucans or inhibit .beta.-glucan synthesis. This strategy has led to the hypothesis of a complex pathway of synthesis and incorporation of (1,6).beta.-glucan into the cell wall in Saccharomyces cerevisiae, as described by Brown et al. in Genetics, Vol. 133, (1993) pp.837-849. In addition, mutation of one killer-toxin-resistance gene affects both (1,6).beta.-glucan and (1,3).beta.-glucan syntheses. Two genes affecting (1,3).beta.-glucan synthesis were isolated using a killer toxin that inhibits (1,3).beta.-glucan synthesis.
Another strategy used in the prior art to isolate genes involved in .beta.-glucan synthesis has been to screen for mutants with altered morphology that require osmotic support for growth. Two Aspergillus nidulans mutants with reduced amounts of cell wall (1,3).beta.-glucan were isolated according to the procedure of Borgia, P. T. & Dodge, C. L. described in J. Bacteriol, Vol. 174 (1992) 377-383. Two groups of osmotic remedial mutants of Schizosaccharomyces pombe having reduced levels of (1,3).beta.-glucan synthase activity were identified. One of these (1,3).beta.-glucan synthase mutants was shown to have a defective .beta. subunit of geranylgeranyltransferase type I. See Diaz, M.; Sanchez, Y.; Bennett, T.; Sun, C. R.; Godoy,C.; Tamanoi,F., Duran, A.& Perez, P. (1993), EMBO J. 12, 5245-5254. Although there has been some success in the isolation and characterization of genes involved in cell wall .beta.-glucan synthesis, analysis of these genes, especially those implicated in (1,3).beta.-linked glucan synthesis, has not resulted in a unifying model of polymer synthesis and cell-wall formation.
It can be seen from the foregoing that a detailed characterization of the (1,3).beta.-glucan synthase complex is needed, along with the characterization of a (1,3).beta.-glucan synthase genes and various antisense constructs against the gene. This basic information and understanding of (1,3).beta.-glucan synthesis and its regulation, subsequent cell-wall assembly and resulting growth and development of fungi is essential to the design and discovery of novel antifungal antibiotics.